Monoquant, an Australian company spun out of Flinders University to commercialize molecular methods to monitor minimal residual disease in blood cancers, has developed a PCR-based DNA assay that can routinely detect one cancerous cell in 10 million normal cells in chronic myeloid leukemia patients, according to a company founder.
The company believes that its assay could be a viable alternative to the current standard method of monitoring minimal residual disease by assaying RNA to detect BCR-ABL translocations.
To that end, researchers from the company and Flinders University are now attempting to push the limit of detection of their assay and minimize potential variations in amplification efficiency using both their proprietary technology, called sequential hybrid primer PCR, and Bio-Rad's QX100 Droplet Digital PCR system.
The Flinders University group and Monoquant are led by Alec Morley, a Flinders professor who along with colleagues in the early 1990s was the first to describe so-called limiting dilution PCR — a precursor of sorts to modern digital PCR — in which DNA samples are diluted so that only one or a few copies are present per well and subsequently PCR amplified.
Morley and his team were able to use this technique to detect as little as two copies of leukemic DNA in a background of 160,000 wild-type DNA molecules, and subsequently showed how the technique could be used to measure response to treatment in patients.
Then, as technology evolved, Morley and colleagues used real-time PCR methods to become one of the first groups to describe RNA-based assays to detect BCR-ABL translocations in CML cells, setting the stage for the current standard of monitoring minimal residual disease.
"Another unique target seemed to be immunoglobulin and T-cell rearrangements in leukemia," Morley recently told PCR Insider. "That was a major thrust of ours for many years, and we've been trying to develop more and more sensitive methods for quantifying that. We originally used limiting dilution PCR, and of course the technology was very crude back then — it had to be done manually. And so when real-time PCR came along, we switched to that."
More recently, Morley and colleagues turned their attention back to CML, and particularly new methods for monitoring minimal residual disease.
"Conventionally it is monitored using RNA," Morley said. "We set out to see if one could monitor it using DNA, which has potential advantages; particularly, RNA is unstable, and [research] has shown that monitoring using DNA is actually a good deal more sensitive."
A major challenge that the group faced, however, is that BCR-ABL translocations are different from patient to patient, and it is difficult to isolate and sequence translocation breakpoints in each case. In order to do this rapidly and efficiently, Morley's group developed sequential hybrid primer PCR for "gene walking." In this method, an initial PCR round using a gene-specific forward primer and a tagged reverse degenerate primer is followed by two or three PCR rounds, each of which uses a gene-specific forward primer and a specially designed hybrid primer.
Since the invention of that technique, Monoquant and Flinders have developed a library of pre-synthesized and pre-tested primers. "Now, when a patient comes in, we sequence the breakpoint, pick the primers off the shelf, and retest them again on that patient … but they virtually always work," Morley said.
Using this method, the researchers "routinely detect down to about 1 in 10 million cells" and, in recent studies, have been able to push that limit even farther — the subject of a pending publication, Morley said.
"We're not sure where that's going to be clinically important," he added. "But there is a lot of interest now in treatment of CML … with new drugs, [where] a substantial minority of patients is getting down to [a point] where the disease is not detectable by RT-PCR." And at that point, in many cases, treatment can be stopped — which is desirable because the drugs cost a lot and cause undesirable side effects.
"It's possible that DNA PCR may give you a much better handle on what's going on in this black zone, and we may be able to improve treatment with a more sensitive technique," Morley said. "But that's speculative at this stage. That's what we think the potential commercial advantage might be."
One potential problem with this approach, however, is that it uses patient-specific primers that may vary in amplification efficiency. "We actually don't find that to be much of a problem, but we thought that down the road the [US Food and Drug Administration] might find it a problem," Morley said.
As such, the Monoquant and Flinders group identified digital PCR as a possible solution — specifically, Bio-Rad's QX100 Droplet Digital platform, which has shown the ability to detect extremely low numbers of mutant DNA in extremely large backgrounds of wild-type DNA.
"We don't know if we can get down to the same level yet using digital PCR," he said. "I think sensitivity is probably going to be determined by the amount of DNA you can obtain." But, he added, it's possible that amplification using Monoquant's patient-specific primer approach may vary from patient to patient, "and digital PCR is not as sensitive to variations in efficiency, because it's essentially an endpoint determination," Morley said.
In addition, "the FDA has, I believe, difficulty in accepting tests based on patient-specific primers," he added. "Whether that will prove to be an obstacle in the long run, I don't know, because there are some situations where you have to use patient-specific PCR, not only in CML."
If the assay is eventually commercialized, it will likely be brought to market by San Diego-based molecular diagnostics firm Invivoscribe, which has a longstanding relationship with Monoquant and Flinders University, and has an exclusive worldwide license to hematopathology IP emanating from those entities.
Invivoscribe has marketed some assays for diagnosing and monitoring lymphoid leukemias or lymphomas based on gene rearrangement IP licensed from Morley's group several years ago, but it is unclear which recently licensed technologies the company is closest to commercializing. Invivoscribe did not respond to emails seeking comment.
However, according to Morley, Invivoscribe has recently been particularly interested in two innovations from Monoquant. "One is … IP related to detecting immunoglobulin T-cell receptor gene rearrangements. [Invivoscribe is] looking at sub-licensing that. With CML, they're also looking at adapting our methodology to next-gen sequencing, but that's further down the track."
Whether the new CML assay from Monoquant reaches market in the US appears to be in the hands of Invivoscribe, although Morley's group continues to refine the test. In the meantime, Flinders does offer a small testing service in Australia that involves isolating and sequencing the BCR-ABL breakpoint, providing primer pairs, and quantifying minimal residual disease for external CML samples.
"We are not really producing a product," Morley said. "We do provide a small service, and that might grow; we'll wait and see. We are not trying to expand this service aggressively. Our main aim is to do the science, publish the science, and push the IP and license it."
On that front, Monoquant owns other IP that may not necessarily be of interest to Invivoscribe, but which Morley believes could prove useful to other laboratories and companies in the molecular diagnostics space.
For instance, it has developed a PCR-based method for quantifying DNA and RNA degradation, which is a common issue in molecular diagnostic assay development.
"Simply by taking a reference gene, and amplifying and determining the Ct of a number of different lengths of that reference gene, from the Ct results you can actually obtain a quantitative figure for RNA degradation," Morley said. "You can use this figure to correct your gene expression data for degradation and get a true measure of the number of transcripts you're trying to enumerate."
Further, the group has developed a PCR-based method for switching primers off and on at defined points during the course of a PCR reaction using antisense oligonucleotides. The technique, called antisense PCR, enabled the development of a highly efficient nested qPCR that can be performed in a closed tube, which has the advantages of obviating potential contamination from opening and closing different reaction tubes.
"We're applying it to see if we can produce a multiplex diagnostic test for [sexually transmitted diseases]," Morley said. "And we're using it to look at a parasite in grapevines, to see if we can develop a sensitive test."
"So we've spun out into a few areas, and our belief is that antisense PCR is a platform technology, and if we can publish science on this that is useful … people will pick it up for their own purposes," he added.